靶向大黄鱼抗菌肽hepcidins的microRNAs鉴定与育种应用研究

由变形假单胞菌引起的内脏白点病是限制大黄鱼产业发展的最主要瓶颈之一。而大黄鱼抗病育种是解决该问题的一种有效方法。基因组选育受限于靶性状遗传力，高昂成本等因素，而基于CRISP/Cas9的基因组编辑在水产养殖性状的遗传改良方面具有巨大的潜力。申请人前期研究表明：大黄鱼抗菌肽LcHamp2-5蛋白具有很强的抗菌活性，其活体注射可明显降低变形假单胞菌感染大黄鱼的死亡率，并且abu-miR-15c-5p是一个靶向抑制 LcHamp2-5基因表达的miRNA。申请人拟通过双荧光素酶报告实验进一步明确该miRNA与LcHamp2-5的靶向关系，并通过miRNA过表达和反义核酸技术揭示该miRNA对LcHamp2-5调控作用及其在大黄鱼抗变形假单胞菌中的功能，并利用申请人建立的大黄鱼基因组编辑技术敲除该miRNA，获得miRNA敲除的大黄鱼抗变形假单胞菌品系。该研究可为大黄鱼抗病育种提供有力的技术支撑。

The large yellow croaker (Larimichthys crocea) is an economically important marine species with the highest annual production among the farmed marine fishes in China. However, the aquaculture industry of this species is suffering from heavy financial losses caused by visceral white nodules disease resulting from Pseudomonas plecoglossicida infection. There is thus an urgent need for genetic improvement to ameliorate the disease resistance of this species. Genomic selection is limited by the heritability of the target traits and high cost. Genome editing technologies, which offer new solutions to these limitations, have tremendous potential to expedite sustainable genetic improvement in aquaculture. Our previous researches showed that chemically synthesized LcHamp2-5 protein, a mature antimicrobial peptide from the large yellow croaker, exhibited a strong antibacterial activity. An intraperitoneal injection in vivo of LcHamp2-5 protein can lower the mortality rate of large yellow croaker against P. plecoglossicida infection. Additionally, dual-luciferase report systems suggested that LcHamp2-5 gene is a target of the miRNA of abu-miR-15c-5p. The applicant intends to further confirm the target relationship between abu-miR-15c-5p and LcHamp2-5 gene by dual-luciferase report systems, and explore the expression regulation of miRNA on LcHamp2-5 gene, and the role of the miRNA in resistance to P. plecoglossicida infection using miRNA overexpression and antisense techniques. And finally, we would establish a new breed of the miRNA knockout large yellow croaker with resistance to P. plecoglossicida infection using the CRISPR/Cas9-based genome editing technique established by the applicant in large yellow croaker. This study will provide a strong technical support for large yellow croaker breeding for disease resistance.